Preprocessing

All the cells were annotated. If the cells from the cluster are represented only by cells from the placenta samples, then we assumed that these were placental cells. If the cells from the cluster are represented only by cells from samples with pericytes, then we assumed that these are pericytes. Endothelial cells are represented by cells from all 4 samples and are located furthest away on the UMAP. The remaining cells are considered fibroblasts.

We tried to use tdTomato expression in order to explicitly separate fibroblasts and pericytes. Since we assume that fibroblasts were transfected by the plasmid with tdTomato as well as muscle cells. However, this did not really allow us to distinguish cells. We do not observe a clear localization of tdTomato on UMAP.

Analysis of HUVEC

##        source number of cells
## 1       My.Hu              85
## 2    My.Pr.Hu             336
## 3    Pl.My.Hu              83
## 4 Pl.My.Pr.Hu             123

DE

The CLU gene is known for its cytoprotective function. It has also been shown that in some cases this gene promotes angiogenesis. PLCG2 is a transmembrane signaling enzyme that catalyzes the conversion of 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate to 1D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) using calcium as a cofactor. IP3 and DAG are second messenger molecules important for transmitting signals from growth factor receptors. HSD17B14, are primarily involved in metabolism of steroids at the C17 position and also of other substrates, such as fatty acids, prostaglandins, and xenobiotics. PPM1L, the encoded protein downregulates apoptosis signal-regulating kinase 1, a protein that initiates a signaling cascade that leads to apoptosis when cells are subjected to cytotoxic stresses. DDIT3 plays an essential role in the response to a wide variety of cell stresses and induces cell cycle arrest and apoptosis in response to ER stress. CHAC1 is a negative regulator of Notch signaling pathway. Anillin (ANLN), an actin-binding protein, reportedly plays a vital role in cell proliferation and migration, particularly in cytokinesis. H19 regulates cell division and aging, is low expressed in old cells. The extracellular domain of TMEFF2 interferes with PDGF-AA–stimulated fibroblast proliferation. KIFF11, the function of this gene product includes chromosome positioning, centrosome separation and establishing a bipolar spindle during cell mitosis.

Based on this analysis, we see that placental cells do not change the expression of markers, however, the expression of ANLN genes is observed, which plays a vital role in division and the KIFF11 gene, which is establishing a bipolar spindle during mitosis. So it can be assumed that the addition of placental cells increases the proliferation of endothelial cells.

In the sample with the addition of pericytes, a marker associated with cellular stress (DDIT3) is observed - a larger number of cells express this gene. It also reduces the expression of the H19 gene, which prevents cellular aging of endothelial cells.

In multiculture, the expression of the cytoprotector gene (CLU) and the downregulates apoptosis gene (PPM1L) is observed.

Thus, it seems that the addition of placenta enhances proliferation, while the addition of placenta together with pericytes compensates for the negative effects of the addition of pericytes.

GSEA

Wiki

Reactome

GO

As a result, we see that the addition of placenta has little effect on endothelial cells, only increases proliferation. While pericytes reduce the activity of collagen formation, enrich the antiinflammatory pathways, encrich the WNT and Notch signaling pathways.

Analysis of Pericytes

##        source number of cells
## 1    My.Pr.Hu           10143
## 2 Pl.My.Pr.Hu           10291

DE

Cluster 0 - all markers are transcription factors involved in development processes.

Cluster 1 - genes associated with tyrosine-kinase.

Cluster 2 - genes of cell junctions. EBF2 stimulate differentiation (I am not sure about our case)

Cluster 3 - NMB stimulates vascularization

Cell-cycle score

Clusters do not differ significantly from each other in the distribution of cells by phases of the cell cycle

GSEA

0 cluster

1 cluster

2 cluster

4 Cluster

There are more cells from cluster 0 in the placenta sample. And in the sample without a placenta, cells from clusters 1 and 2 predominate.

In cluster 0, amino acid metabolism is enriched and the “RUNX2_REGULATES_GENES_INVOLVED_IN_CELL_MIGRATION” gene set is enriched. Perhaps the placenta enhances these processes.

In cluster 1, the processes associated with the organization of ECM are more enriched than others, and the enrichment of angiogenesis is also observed. There is also an increased activity of metalloproteinases. This may indicate that the placenta inhibits these processes. However, it is worth noting that cells from this cluster are present in both samples. But there are more of these cells in the sample without the placenta.

By samples

DE

GSEA

Analysis of Fibroblasts

##        source number of cells
## 1       My.Hu           14840
## 2    My.Pr.Hu            1652
## 3    Pl.My.Hu           13054
## 4 Pl.My.Pr.Hu            1050

Based on the distribution of clusters by samples, clusters 3 and 5 (cells grown with the addition of placenta predominate) and clusters 6, 5 and 7 (cells grown with the addition of pericytes predominate) look the most interesting

Cell-cycle score

Cluster 3 is represented by a large number of dividing cells.

This table shows how many cells in each sample are represented by cells of 3 clusters. According to the results of this table, it can be seen that indeed the addition of placenta improves cell proliferation

##        source percentage_of_cluster3
## 1       My.Hu                    7.5
## 2    My.Pr.Hu                   23.8
## 3    Pl.My.Hu                    7.0
## 4 Pl.My.Pr.Hu                   27.2

DE

Markers have been defined for each cluster

GSEA

GSEA analysis was performed to describe the interesting clusters

Cluster 3

Enriched pathways associated with the cell cycle and cell proliferation are observed in cluster 3. There is also a high level of enrichment for cell-cell communications. Also, the activity of the WNT pathway is increased in all clusters of interest (3, 6, 7, 8)

Cluster 6

In cluster 6, there is an increased enrichment of the TP53 signaling pathway associated with the cellular response to stress and complement and coagulation processes. Cluster 6 cells are predominantly represented by cells grown with the addition of pericytes. Perhaps the addition of pericytes increases stress.

Cluster 7

Cluster 7 is characterized by high involvement in the processes of ECM formation, especially laminin interactions. There is an increased activity of the PDGF pathway.

Cluster 8

Inflammatory processes and signaling pathways associated with interferons are increased in cluster 8.

In conclusion, it can be said that the placenta enhances the proliferation of fibroblasts. The addition of pericytes improves the formation of EСM, as well as enhances the PDGF signaling pathway. However, the addition of pericytes also increases the enrichment of inflammatory processes.

Both with the addition of pericytes and placenta, an increase in the enrichment of the WNT signaling pathway was observed.

By samples

GSEA

### DE

Placenta cells

Placenta cells additionaly secrete various ECM proteins that provide structural support and can influence muscle cell behavior.